The present invention relates to the field of diagnostics by means of microscopic sample analysis, and specifically relates to a method and apparatus for preparing thin microscopic samples of liquids, and in particular monolayers of red blood cells.
Microscopic examination of blood films is an important part of the hematologic evaluation. Today, three methods for preparing thin blood films are in use, namely the xe2x80x9cwedge-slide methodxe2x80x9d, the xe2x80x9cspinner methodxe2x80x9d and the xe2x80x9ccoverglass methodxe2x80x9d (see, e.g. xe2x80x9cClinical Diagnosis and Management by Laboratory Methodsxe2x80x9d, Nineteenth Edition, 1996, edited by John B. Henry).
In the xe2x80x9cwedge-slide methodxe2x80x9d, a drop of blood is placed onto a slide that is on a flat surface. A second (spreader) slide is pressed at an angle of 30 to 45 degrees against the first slide and moved along the first slide, which results in the formation of a moderately thin blood film that is then dried in air. The quality of the generated blood smear will strongly depend on the personal skill level of the technician. To overcome the need for highly skilled personnel, both manual apparatus (U.S. Pat. No. 4,494,479 to Drury) and automated apparatus (U.S. Pat. No. 4,407,843 to Sasaki; U.S. Pat. No. 3,683,850 to Grabhorn; U.S. Pat. No. 3,880,111 to Levine et al.; U.S. Pat. No. 4,392,450 to Prevo; WO 9,641,148 to Levine et al.) for executing the wedge-slide method have been proposed.
In addition to being time consuming, the physical action of the spreader slide tends to distort the morphology of many of the cells. In view of this, an alternative method for the preparation of blood samples known as the xe2x80x9cspinner methodxe2x80x9d has been proposed (see, for example, U.S. Pat. No. 5,549,750 to Kelley) wherein a drop of blood is disposed onto a slide which is then spun to create a monolayer of randomly distributed red blood cells (RBCs). It has been found, however, that drying of the red blood cells produces undesirable types of distortions, particularly a loss of central pallor for many of the RBCs as they dry. It is not entirely clear what causes these shape changes, but they apparently are caused by surface tension, charges and/or drying effects. To inhibit cell morphology distortions from occurring during drying, it has been proposed to preserve the morphologies by applying fixing agents after forming the monolayer, but prior to drying (U.S. Pat. No. 4,209,548 to Bacus; U.S. Pat. No. 4,483,882 to Saunders).
The wedge-slide method as well as the spinner method require relatively complex apparatus and involve time consuming procedures. A simpler way to produce blood films is the xe2x80x9ccoverglass methodxe2x80x9d where two quadratic coverglasses are being used. A first glass, with a drop of blood attached to the center of the underside, is placed crosswise on a second glass so that the corners appear as an eight-pointed star. If the drop is not too large and if the glasses are perfectly clean, the blood will spread out evenly and quickly in a thin layer between the two surfaces. After spreading stops, the two glasses are pulled apart on a plane parallel to their surfaces. The two blood films are then dried in air.
While the coverglass method does not require auxiliary apparatus, the quality of the blood smears will again depend strongly on the skill level of the technician performing the procedure. Moreover, executing the method includes increased risk because the thin pieces of glass that contain the blood sample may break during the separation step. And, finally, drying the blood films may cause changes in the cell morphology.
It has been suggested, in U.S. Ser. No. 09/085,851, now U.S. Pat. No. 6,180,314, issued on Jan. 30, 2001 to produce thin liquid samples for microscopic analysis by (1) arranging spacers on a microscope slide outside of the slide""s center, by (2) disposing a drop of blood onto the microscope slide near to its center, by (3) positioning a flexible coverglass onto the spacers, by (4) applying a downward force to the center of the flexible coverglass so that the coverglass touches the blood, and by (5) suspending the application of said force. In the moment the coverglass is touching the drop of blood, the blood is spreading outwards and adhesion forces hold the flexible coverglass down so that a very thin layer of liquid is formed.
While this method provides thin layers of blood, and in particular monolayers of isolated red blood cells, it is still quite labor-intensive. More specifically, the positioning of the flexible coverglass onto the spacers requires concentration from the operator. There are also concerns that the thin coverglass may break during operation, and the possibility that the operator may be injured. Moreover, the application of the downward force requires some degree of training.
For the production of monolayers of red blood cells, it has also been suggested (S. Wardlaw, xe2x80x9cAnalysis of quiescent anticoagulated whole blood samplesxe2x80x9d, U.S. Ser. No. 09/249,721, now U.S. Pat. No. 6,235,536, issued on May 22, 2001 and S. Wardlaw, xe2x80x9cCalibration of a whole blood sample analyzerxe2x80x9d, U.S. Ser. No. 09/248,135, now U.S. Pat. No. 6,127,184, issued on Oct. 3, 2000) to design a cuvette-like optical sample container for the cell suspension that has different optical pathlengths in different areas. In at least one area, the thickness of the liquid layer of un-diluted blood is so thin (2 to 7 microns) that monolayers of isolated RBCs are formed. In another region, the liquid layer is thicker (7 to 40 microns), and typical chain-like aggregates of RBCs (xe2x80x9cRoleauxxe2x80x9d) are forming. The thick area is used to determine the hematocrit (HCT), and the thin area is used to determine the volume of single red blood cells (RCV). As has been found by the inventors of the present invention, the formation of a monolayer of red blood cells in a cuvette having areas of different thickness according to prior art is often impaired and may not be reliable.
The use of compartments of different thickness in the prior art is necessary for allowing the determination of the RCV and the HCT. The HCT can not be measured within the monolayer region, and the RCV can not be determined within the Roleaux region. RCV and HCT are usually determined employing the principle of fluorescence volume exclusion. In this method, the plasma is stained with a fluorescent dye, and the RCV as well as the HCT are measured by determining the partial volume that is excluded from emitting fluorescence by the RBCs. The principle relies on homogeneous excitation intensity throughout the whole sample volume, and also on homogeneous photon collection efficiency throughout the whole sample volume. FIG. 1 shows a plot of the excitation intensity along the z-axis within a microscope, assuming an objective lens with a numerical aperture, NA=0.4, and an excitation center-wavelength of 500 nm. A similar situation applies to the photon collection efficiency through the objective lens. Due to the lack of homogeneous excitation and homogeneous photon collection, the determination of the HCT in a cuvette of thickness between 15 and 40 xcexcm is somewhat problematic.
In view of the above problems there is a need for a reliable way to produce monolayers of isolated RBCs. There is also a need for a method for determining the HCT of whole blood in a cuvette having a height below 10 xcexcm.
It is an objective of the present invention to provide a method and apparatus to produce thin samples of liquids for microscopic analysis, and in particular thin samples of whole blood, whereby monolayers of isolated red blood cells are formed, and whereby the undiluted sample""s HCT can be determined from a measurement within the monolayer.
According to the present invention, the above objective is achieved with an optical cuvette with at least one transparent window, whereby said cuvette has a thickness below a certain value, whereby the interior of the cuvette is separated into a plurality of successive compartments by means of separating walls, whereby each separating wall comprises a first type of through-channel allowing RBCs and blood plasma to flow through, and whereby each separating wall comprises also a second type of through-channel allowing only blood plasma to flow through, the channels being designed in a way that a significant percentage of the RBCs arriving at a separating wall can pass through, so that the particle dilution per separating wall is relatively low, whereby the liquid sample enters the first of the compartments through an entrance, where there are small venting openings in the compartments to allow for air venting during filling, and whereby characteristic parameters of the blood sample entering the cuvette are determined by analyzing the monolayer of isolated RBCs that forms within the last compartment, using the principles of fluorescence volume exclusion and/or absorption volume exclusion in connection with a microscope.
According to the present invention, the thickness of the cuvette should be so small that no Roleaux formation develops within the one compartment that is farthest away from the entrance. A typical device according to the present invention has a height preferably between 3 and 10 xcexcm, comprises preferably at least five separating walls, whereby each of the separating walls allows one half of the arriving RBCs to pass through. This results in an overall xe2x80x9cdilutionxe2x80x9d allowing for the formation of monolayers of isolated RBCs in the compartment farthest away from the entrance. The HCT value of the sample entering the cuvette can be determined from a measurement of the HCT value within the last compartment.